Membrane-enclosed biological structures can support a voltage difference between the inside and the outside of the membrane. This voltage, also called a membrane potential, serves a variety of biological functions, including carrying information (e.g., in neurons), acting as an intermediate in the production of ATP (e.g., in bacteria and mitochondria), powering the flagellar motor (e.g., in bacteria), and controlling the transport of nutrients, toxins, and signaling molecules across the cell membrane (in bacteria and eukaryotic cells).
In spite of its fundamental biological role, membrane potential is very difficult to measure. Electrophysiology involves positioning electrodes on both sides of the membrane to record voltage directly. Electrophysiological experiments are slow to set up, can only be performed on one or a few cells at a time, cannot access deeply buried tissues (e.g., in vivo), do not work for cells that are too small (e.g. bacteria) or are enclosed in a hard cell wall (e.g. yeast), or are motile (e.g., sperm), cannot be applied to long-term measurements, and usually damage or kill the cell under study. Accordingly, novel methods for measuring membrane potential are needed.
To disentangle the complex interactions underlying neural dynamics, one would like to visualize membrane voltage across spatial scales, from single dendritic spines to large numbers of interacting neurons, while delivering spatially and temporally precise stimuli.1,2 Optical methods for simultaneous perturbation and measurement of membrane potential could achieve this goal.3 Genetic targeting of the stimulation and recording to genetically specified cells is useful in intact tissue where closely spaced cells often perform distinct functions. Genetic targeting in vitro is also useful for characterizing heterogeneous cultures that arise during stem cell differentiation to neurons,4 or while studying neurons co-cultured with other cell types.
Optical stimulation has been demonstrated with glutamate uncaging,5 photoactivated ion channel agonists6, and microbial rhodopsin actuators.7,8 Genetically encoded functional readouts include reporters of intracellular Ca2+ and membrane voltage.9-14 Voltage-sensitive dyes offer good speed, sensitivity, and spectral tuning,15,16 but cannot be delivered to a genetically specified subset of cells and often suffer from phototoxicity.
Simultaneous optical stimulation and readout of neural activity have been implemented via several combinations of the above techniques.17-21 However, robust genetically targeted all-optical electrophysiology has not been achieved due to limitations on the speed and sensitivity of genetically encoded voltage indicators (GEVIs), and spectral overlap between existing GEVIs and optogenetic actuators. GFP-based GEVIs experience severe optical crosstalk with even the most red-shifted channel rhodopsins, which retain ˜20% activation with blue light excitation.22 Therefore, there remains a need for sensitive, fast, and spectrally orthogonal tools for genetically targeted simultaneous optical perturbation and measurement of membrane voltage.